PROJECT SUMMARY AND ABSTRACT There is a great need for new treatments for Inflammatory Bowel Disease (IBD). IBD affects over 1% of US adults, leading to disability, hospitalization, and surgery. IBD is characterized by damage to the ?lining? of the intestine, also known as the intestinal epithelium. Intestinal epithelial damage leads to inflammation, bleeding, and an increased risk of colon cancer. Since a damaged intestinal epithelium is the defining feature of IBD, we want to understand what genes preserve intestinal epithelial integrity in order to better treat IBD. Genome-wide association studies (GWAS) have linked hundreds of genes with small individual effects to IBD, but it is unclear how these genes cooperate to increase susceptibility to intestinal injury and inflammation. We have a mouse model to study the role of two IBD-associated genes, A20 and ABIN-1, in intestinal epithelial cells (IECs). We were the first to show that A20 and ABIN-1 work together to maintain a healthy intestinal epithelium by preventing IEC injury. We grew intestinal organoids from the intestines of these mice. Intestinal organoids are miniature replicas of the intestinal epithelium grown in a dish in the lab. Using this system, we discovered that IECs lacking A20 and ABIN-1 are exquisitely sensitive to cell death from tumor necrosis factor (TNF). TNF is one of the primary death signals in the intestine, and antibodies against TNF are commonly used to treat IBD. We have dramatic evidence that A20 and ABIN-1 cooperate to preserve intestinal well-being, but we still need to understand the specific ways in which they preserve IEC health. We will use the mouse model and our organoids to determine which inflammatory cells injure IECs in the absence of TNF. We have evidence that inflammatory cells secrete a factor or factors other than TNF that kill A20 & ABIN-1 deficient IECs. This could be a very important clue as to why some patients with IBD do not respond to anti- TNF neutralizing antibodies. Additionally, A20 and ABIN-1 might cause epithelial injury by altering how intestinal stem cells ?differentiate? or mature into other IEC subsets. Two subsets of particular interest are paneth cells and goblet cells, but there are many other subsets that we will study using our unique system. Finally, and perhaps most importantly, we will also grow intestinal organoids from IBD and non-IBD patients and test susceptibility to cell death. Using the organoids, we can attempt to rescue cell death with small molecules and drugs that are currently in clinical trials. We will analyze IEC subsets in the organoids, and see how TNF and other stimuli perturb IEC subsets. Lastly, we will work on ?editing? human organoids with a technique called CRISPR/Cas9, to improve our ability to study other genes in IBD. The long-term goal of these studies is to identify approaches of treating IBD that protect epithelial cells from injury, rather than simply suppressing the immune system.